Deck 21: Biomolecule Purification

Question 1

If a protein binds a specific ligand

A) that ligand may be used to purify the protein in vitro
B) the ligand may interfere with studying the protein in situ
C) the ligand may interfere with protein folding
D) Both A and B
E) All of the above

Answer 1

Both A and B

Question 2

Large quantities of proteins may be isolated from

A) E.coli
B) yeast
C) plant tissue
D) animal tissue
E) All of the above

Answer 2

All of the above

Question 3

Which is a disadvantage to using bacteria as a source of proteins for purification?

A) Molecular biology techniques can be used to overexpress the protein in these cells
B) Proteins can be extracted from the cells themselves, or from the media in which the cells are grown
C) differences in post-transcriptional modifications may occur
D) They grow more quickly than plant or animal tissue
E) There are fewer ethical issues involved in using bacterial cells for experimentation

Answer 3

differences in post-transcriptional modifications may occur

Question 4

Proteins used as drugs are called ___.

Question 5

Which is NOT a protein molecule used as a pharmaceutical?

A) insulin
B) Celestone (a corticosteroid)
C) EPO (erythropoietin)
D) Enbrel (a monoclonal antibody)
E) Remicade (a monoclonal antibody)

Question 6

To yield sufficient biologics, pharmaceutical companies use ___ to grow sufficient cells, commonly with ____ versions of natural proteins.

Question 7

One problem with producing biologics in cells that differ from human cells is that proteins may be alternately ___, which could affect purification and/or function of the ultimate pharmaceutical.

A) phosphorylated
B) glycosylated
C) cleaved
D) processed
E) All of the above

Question 8

As protein purification proceeds, the number of contaminants increases while the sample is enriched with the desired protein. The purity of the protein is assessed based on "fold purification."

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 9

A typical protein purification plan might maximally enrich a sample ___ fold above that found in the original cell lysate.

Question 10

At each step of a protein purification scheme, purity should be ascertained by, for example,
I. SDS-PAGE gel electrophoresis
II. specific activity of antibodies
III. western blotting
IV. volume measurement

A) I only
B) IV only
C) I, II and III
D) II, III and IV
E) I, III and IV

Question 11

A researcher is purifying a structural protein in ovaries. Which would NOT be useful information for a protein purification table?

A) volume (mL)
B) protein (mg)
C) activity (units)
D) purification (fold)
E) yield (%)

Question 12

As a successful purification scheme is carried out, the specific activity of a desired enzyme may decrease, though the samples are enriched in the desired enzyme. This is because specific activity is calculated per mg of protein, and often mg protein decreases as the scheme progresses.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 13

Cellular lysis

A) is necessary if the desired protein is released to the media in which cells are grown
B) can be accomplished by homogenizing with a centrifuge
C) can be accomplished by sonicating the cells with UVB waves
D) can be accomplished by using a blender to break open the cells
E) All of the above

Question 14

Once cells have been lysed, the next step in protein purification commonly includes

A) low speed centrifugation
B) high speed ultracentrifugation
C) sucrose gradient centrifugation
D) ion exchange chromatography
E) salting out

Question 15

High speed ultracentrifugation

A) allows separation of proteins of varied molecular weights, but only if the proteins in the mixture differ by >50,000 MW
B) allows separation of proteins of varied molecular weights, but only if the proteins in the mixture differ by >5,000 MW
C) allows separation of proteins of varied molecular weights, but only if the proteins in the mixture differ by >100,000 MW
D) does not allow separation of individual proteins
E) is commonly used to separate positively-charged proteins from negatively-charged proteins

Question 16

By increasing the salt concentration in a solution, proteins

A) are somewhat more soluble
B) may become very soluble
C) may show decreased solubility
D) Both A and B
E) All of the above

Question 17

Addition of low salt concentrations to a protein-containing solution,

A) hydrophobic interactions among protein molecules increase
B) solubility of proteins in solution decreases
C) the solution has relatively low ionic strength
D) Both A and B
E) All of the above

Question 18

By adding increasing amounts of salt to a solution containing proteins,
I. the salt causes waters of hydration to form around individual protein
Molecules
II. salting in occurs, and protein solubility increases
III. proteins aggregate at very high salt concentrations
IV. at very high salt concentrations, salting out occurs, and protein solubility
Increases

A) I and II
B) II and III
C) III and IV
D) I, II and III
E) I, II, III and IV

Question 19

With salting in

A) ammonium (NH4+) salts are commonly used
B) protein denaturation may occur
C) proteins tend to precipitate out of solution
D) less water is available for forming shells of hydration around protein molecules
E) very similar salts and salt concentrations are used for all types of proteins

Question 20

If it is important that protein stability is maintained through a purification scheme, and little denaturation can be tolerated, your scheme should include the use of

A) CO₃^2- or SO₄^2-
B) S₂O₃^2- or Cl-
C) CO₃^2- or NO₃-
D) I- or SCN-
E) H₂PO₄- or F-

Question 21

Dialysis is used to separate small molecules, like salts, from proteins of interest in a purification scheme. The sample is placed in a dialysis bag, which is immersed in buffer, and the salts will separate and remain in the dialysis bag, which can be removed and discarded.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 22

Dialysis tubing or bags

A) are made of cellulosic plastic or fabric
B) have varied pore sizes, called molecular weight cut-offs
C) interact with proteins to cause them to precipitate
D) Both A and B
E) All of the above

Question 23

Ultrafiltration is a combination of

A) ion-exchange and size-exclusion chromatography
B) ion-exchange chromatography and centrifugation
C) size-exclusion chromatography and centrifugation
D) dialysis and centrifugation
E) size-exclusion chromatography and dialysis

Question 24

Chromatography

A) employs columns that are filled with solid supports
B) separates components of a solution as they move at different rates
C) uses resins or beads
D) Both A and B
E) All of the above

Question 25

Which is NOT true of beads used in chromatography?

A) The size of the beads is called the mesh size
B) A larger bead number represents a smaller bead particle size
C) Smaller beads have lower surface area
D) Smaller beads allow only a slower flow of buffer through the column
E) Both A & B

Question 26

High pressure liquid chromatography (HPLC) is not best for separating proteins because

A) it takes too long for high molecular weight molecules to elute
B) only highly acidic buffers can be used which alter proteins' primary structures
C) its required metal tubing interferes with protein purification
D) fast protein liquid chromatography (FPLC) runs at higher and more useful pressures
E) it cannot be accomplished in the cold

Question 27

To monitor the protein concentration of samples eluted during column chromatography, ___ is commonly used, based on the presence of the amino acids such as ___ in the eluted proteins.

A) UV absorbance; phenylalanine
B) UV absorbance; alanine
C) fluorescence; alanine
D) NMR; phenylalanine
E) NMR; alanine

Question 28

A protein rich in tryptophan would be expected to have high spectrophotometric absorbance in the range of

A) 20-40 nm
B) 80-120 nm
C) 150-200 nm
D) 270-300 nm
E) 350-400 nm

Question 29

To facilitate quick analysis of eluents from a chromatographic column, a ___ is installed between the column outflow and collecting tubes.

Question 30

Analysis of proteins from the inner mitochondrial membrane which are bound to cytochrome groups could likely be facilitated by

A) UV spectrophotometry
B) visible spectrophotometry
C) flouresence spectrophotometry
D) NMR
E) HPLC

Question 31

Which is NOT important to size-exclusion chromatography?

A) size of pores fond in resin beads
B) mesh size
C) hydrodynamic properties of proteins being separated
D) ionic properties of proteins being separated
E) column geometry

Question 32

In size exclusion chromatography
I. molecules too large to interact with beads elute in the void volume
II. molecules of intermediate size may enter the gel pores and are
Retained to a lesser extent than the largest molecules
III. beads and buffer together are termed the "bed volume"
IV. the elution volume is that at which the targeted protein elutes

A) I and II
B) II and III
C) I, II and III
D) I and IV
E) I, III and IV

Question 33

If the following mixture of proteins was applied to a size-exclusion chromatography column, what would be the order of elution?
Proteins with molecular weights: myoglobin (17.7 kDa), hemoglobin (64.5 kDa), lysozyme (14.3 kDa) and triose phosphate isomerase (57.4 kDa)

A) lysozyme, myoglobin, triose phosphate isomerase, hemoglobin
B) triose phosphate isomerase, hemoglobin, lysozyme, myoglobin
C) hemoglobin, myoglobin, lysozyme, triose phosphate isomerase
D) hemoglobin, triose phosphate isomerase, myoglobin, lysozyme,
E) cannot be determined

Question 34

In size exclusion chromatography, resins with small pore sizes will be able to resolve large molecules but not small ones. Therefore, the size of the pore must be matched to the estimated size of the protein to be purified.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 35

Longer size exclusion chromatography columns

A) may be 1-2 m in length
B) generally have lower flow rate
C) likely will limit the diffusion of the protein of interest and decrease resolution
D) may cause high pressures that might crush the resin
E) Both A and B

Question 36

Which is NOT included among the hydrodynamic properties of particles such as proteins in aqueous solution?

A) viscosity
B) sedimentation coefficient
C) diffusion coefficient
D) secondary structure
E) hydrodynamic radius

Question 37

The hydrodynamic radius may complicate the planned elution of a protein of a particular MW in that

A) a protein with spherical shape may elute later than expected from a column packed with 25-50 size beads
B) a protein with a prolate shape may elute later than expected from a column packed with 25-50 size beads
C) a protein with a prolate shape may elute earlier than expected from a column packed with 25-50 size beads
D) a protein with oblate shape may elute earlier than expected from a column packed with 1200 size beads
E) Both A and B

Question 38

Hydrogen bonds are strongest when

A) an electropositive atom is attracted to a polarized hydrogen attached to -OH or -NH
B) an electronegative atom is attracted to a polarized hydrogen attached to a phenyl group
C) all groups are arranged in a linear fashion
D) a bond length of 50 angstroms can be made
E) All of the above

Question 39

London dispersion forces, a type of van der Waals interaction, are much weaker than hydrogen bonds. The strength of London dispersion forces varies with the inverse square of the distance between two charged species held within a molecule.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 40

Dipole-dipole interactions

A) may occur between two groups holding opposite partial charges
B) are another name for salt bridges
C) are optimized at rather long distances
D) are the predominant weak intermolecular force in hydrocarbons
E) are optimized by more polar solvents

Question 41

Resins used in affinity chromatography commonly form weak interactions with atoms important to the ___ structure of proteins.

A) quarternary
B) tertiary
C) primary
D) Both A and B
E) All of the above

Question 42

Sepharose resin modified with a butyl or phenyl group would be used for

A) size exclusion-chromatography
B) hydrophobic interaction chromatography
C) ion-exchange chromatography
D) high pressure liquid chromatography
E) salt-bridge chromatography

Question 43

A C-8 column would preferentially bind

A) strongly hydrophobic proteins
B) proteins enriched in tyrosine and phenylalanine
C) negatively charged proteins
D) proteins with low MW
E) Both A and B

Question 44

A protein mixture is commonly applied to an HIC column under high ___ conditions, and the protein of interest is eluted with buffer of decreasing ____.

A) salt; ionic strength
B) cation; ionic strength
C) acid; acidity
D) basic; acidity
E) salt; acidity

Question 45

Gradients allow a gradual change in buffer strength, and are convenient, as gradient mixers or pumps can be automated. Gradients are often used for eluting buffers.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 46

How does reverse-phase chromatography differ from HIC?

A) In HIC, eluting buffers can be gentler
B) In HIC, the hydrophobic interactions between proteins and resin are weaker
C) In HIC, the hydrophobic interactions between proteins and resin are stronger
D) In reverse-phase, low MW proteins elute from the column earliest
E) Both A and B

Question 47

Ion exchange chromatography
I. separates proteins from a mixture based on each protein's isoelectric point
II. depends on the pH at which a protein is electronically neutral
III. depends on the pH at which the positively charged residues of a protein
Equal the negatively charged residues
IV. uses coulombic effects to separate proteins

A) I only
B) I and II
C) IV only
D) I, II and IV
E) I, II, III and IV

Question 48

Which is NOT true of a cellular protein with an acidic isoelectric point?

A) Its pI (isoelectric point) is likely at pH 1 or 2
B) It contains more anionic amino acid residues than cationic amino acid residues
C) At neutral pH, its carboxyl groups would be deprotonated
D) It would bind to a positively charged ion-exchange resin
E) It would be best to use an anion exchange resin to separate this cellular protein

Question 49

Carboxymethyl (CM) ion-exchange resin

A) bears a positive charge
B) contains amino groups, including newer quarternary amines
C) is termed an anion-exchange resin
D) binds proteins relatively weakly due to their low charge density
E) None of the above

Question 50

A researcher adds a "tag" of several histidine residues to an engineered protein in order to purify the protein using

A) size-exclusion chromatography
B) MAC
C) an immunoaffinity column
D) a cation exchange column
E) HIC

Question 51

Metal affinity chromatography (MAC) commonly uses divalent anions which are bound to a resin. MAC is useful when a protein of interest has an aspartate residue on its surface, which can bind the metal column packing.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 52

Like ion exchange chromatography, MAC

A) eluent buffer is an imidazole salt
B) eluent buffer contains high concentrations of histidine
C) eluent buffer competes with bound protein for resin binding sites
D) depends on increased pH to elute the bound protein
E) never uses a gradient eluent buffer

Question 53

Biotin's cellular function includes

A) fatty acid biosynthesis
B) gluconeogenesis
C) binding circulating lipids
D) electron transport in mitochondrial inner membrane
E) Both A and B

Question 54

Which is NOT true of biotin-avidin interaction?

A) It has a very small dissociation constant
B) It mimics enzyme-substrate or receptor-ligand interactions
C) It has very high affinity
D) The interaction is covalent in nature
E) It likely is important to inhibit bacterial growth

Question 55

A researcher biotinylates a protein; this protein can then be purified by passing it through a column packed with resin coated with

A) avidin
B) divalent metal cations
C) DEAE (diethylaminoethyl groups)
D) phenyl groups
E) IgG

Question 56

A biotinylated protein likely will not be denatured or modified too drastically, so may be reintroduced to a cell. If the biotinylated protein binds other cellular molecules, avidin may be used to purify the biotinylated protein paired to its partner.

A) Both sentences are true
B) Both sentences are false
C) The first sentence is true and the second sentence is false
D) The first sentence is false and the second sentence is true

Question 57

The most common immunoaffinity column packing is with

A) IgG
B) IgA
C) IgM
D) IgE
E) IgD